ANTI-ZONA PELLUCIDA ANTIBODIES FOR DELIVERY OF THERAPEUTIC AGENTS TO THE OVARY

Abstract: The immunochemical agents for specifically targeting a therapeutic agent to the ovary includes an anti-zona pellucida antibody conjugated to a therapeutic agent.

Description: TITLE: ANTI-ZONA PELLUCIDA ANTIBODIES FOR DELIVERY OF THERAPEUTIC AGENTS TO THE OVARY FIELD OF THE INVENTION This invention relates to antibodies which specifically bind to zona pellucida and to immuno- chemical agents made from those antibodies, and to therapeutic methods utilizing such immunochemical agents. BACKGROUND OF THE INVENTION Ovarian disease, and particularly ovarian cancer is preferably treated by agents which are specifically targeted to the ovary. In addition, agents for fertility control may be more effective when such agents are specifically directed and distributed to the ovary and not diluted in other, non-ovarian tissues. The mammalian zona pellucida surrounds the oocytes present within the ovary. Its unique structure includes a novel glycoprotein network. Because of the unique nature of the zona pellucida proteins, antibodies raised against these proteins are generally specific to the ovary. Thus, anti-zona pellucida antibodies are ideally suited as carriers for targeting therapeutic agents to the ovary. Cytotoxic agents have been conjugated to ovarian tumor cells and specific tumor cell antigens for the purpose of targeting delivery of the cytotoxic agent to tumor tissues (See, for example, U.S. Patent Nos. 4,956,453 to Bjorn et al; 4,806,494 to Pastan et al; and 4,925,922 to Byers et al) . Delivery of the agent to the ovary requires that the ovarian tissue contain the specific tumor antigen recognized by the antibody. While such antibody recognition may be expected when the antibody is raised against the patient's own tumor cells, tumor antigens vary from one patient to another and a single antibody may not be expected to carry cytotoxic agents to the ovaries of all patients. In addition, non-carcinogenic conditions of the ovary would benefit from targeted delivery of therapeutic agents. Targeted, specific delivery of therapeutic agents is desired to enable lower dosages of therapeutic agents and specific ovarian action of non- specific agents. Targeted, specific delivery of therapeutic agents is desired to enable use of lower dosages of therapeutic agents, especially those which may be detrimental to normal tissues, and to permit specific ovarian action of non-specific agents. It would be of great utility to provide antibodies for use in specifically targeting therapeutic agents t,o the ovary, which antibodies did not rely on the recognition of antigens which vary from patient to patient. SUMMARY OF THE INVENTION Monoclonal and polyclonal antibodies raised against zona pellucida antigens have been found to carry a desired therapeutic agent specifically to the ovary without significant loss of the therapeutic agent to non-ovarian tissues. Such antibodies are used to specifically direct therapeutic agents to the ovary without significant variation in recognition from ovary to ovary. In a preferred embodiment, anti-zona pellucida antibodies target agents to the ovary for specific fertility and contraceptive effects. DETAILED DESCRIPTION OF THE INVENTION Anti-zona pellucida antibodies include those antibodies raised against whole or fractionated mammalian zona pellucida and those raised against specific zona pellucida proteins, native or recombinant, or fractions thereof. The antibodies may be raised against any species of mammalian zona pellucida, although non-rodent species are preferred. It has been reported that non-rodent zona pellucida antibodies may not be cross-reactive with rodent zona pellucida, but are cross-reactive among non-rodent species. Antibodies, monoclonal and polyclonal, may be prepared by methods generally known in the field. In general, polyclonal antibodies are raised in host animals by the injection of zona pellucida antigen into a host animal. The host animal's blood serum is removed, and fractionated to obtain antisera. The antisera is screened for the presence of anti-zona pellucida antibodies by demonstrated reactivity with zona pellucida and zona pellucida antigens, e.g., by known immunoassay techniques. The antibodies are also screened for specificity to ovarian tissue by methods known in the art, e.g., immunocytochemistry or Western Blot analysis. Monoclonal antibodies are produced from hybridoma cells by known methods in the art. In general, a host animal is injected intraperitoneally with zona pellucida antigen. The spleens of immunized animals are collected and used to prepare hybridomas with a murine tumor partner using the general zymotic cell hybridization technique of Kohler and Milstein, Nature, 1975, 256:495-497. This technique involves fusing tumor cells and splenocytes using a fusogen such as polyethylene glycol. After fusion, the hybrid cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells. The hybridomas are expanded, if desired, and conditional culture medium is assayed for anti-zona pellucida activity by conventional immunoassay procedures including radioimmunoassay, enzyme immunoassay or fluorescence immunoassay. Hybridomas that produce monoclonal antibodies may be grown in vitro or in vivo using known procedures. Preferably the hybridomas are maintained as ascites fluid in mice. The monoclonal antibodies may be isolated from culture media or body fluids by conventional immunoglobulin purification procedures such ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration if desired. Important characteristics of the monoclonal and polyclonal anti-zona pellucida antibodies of the present invention include their reactivity with zona pellucida antigen and their specificity to ovarian tissues. The antibodies of the present invention may be raised against a zona pellucida antigen of the same species as the species to be treated (self-antigen) , or may be raised against a second species zona pellucida (foreign antigen) , where species show antigenic cross reactivities. For example, polyclonal antisera produced against a first species zona pellucida in a second host species may recognize the first, second, and additional species zona pellucida as tested by Western blot, ELISA, or immunohistochemical techniques. Antibodies may be labeled or conjugated to therapeutic agents for use in the specific delivery of such agents to the ovaries of the first, second or other species. Similarly, recombinant zona pellucida proteins (foreign or self-antigens) and synthetically produced zona pellucida peptides may be used as antigens to produce antibodies useful in the method invention. Anti-zona pellucida antibodies are conjugated to chemical therapeutic agents by known methods in order to form a desired immunochemical agent. The conjugation may be made using a variety of bifunctional protein coupling agents. Examples of such reagents are N-succinimidyl-3-(2-pyridyldithio) propionate, iminothiolane, bifunctional derivatives of i ido esters, aldehydes such as glutaraldehyde, bis-azido compounds such as bis(P-diazonium benzoyl) -ethylene diamine, diisocyanates such as tolylene 2,6- diisocyanate, and bis-active fluorene compounds such as 1,5-difluoro-2,4-dinitrobenzene. Examples of desired therapeutic agents for coupling to the anti-zona pellucida antibodies include radioisotopes, chemotherapy agents, cytotoxins, and the like. Radioisotopes are preferably high linear energy emitting isotopes, e.g., Y, Pr, and the like. The cytotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial, fungal, or plant origin. Examples of such enzymatically active toxins are diphtheria toxin A, exotoxin A, Ricin A, abrin A, modeccin A, alpha sarcin, curcin, crotin, restrictocin, phenomycin, and enomycin. The preferred cytotoxins are ribosomal inhibiting proteins such as the A chain of ricin or the A chain of abrin. The A chain of ricin has been employed in cytotoxic conjugates as disclosed in U.S. Patent No. 4,590,071. The therapeutic immunochemical agents of the present invention may be used in a variety of therapeutic applications where specific, targeted delivery of a therapeutic agent to the ovary is desired. Examples of such therapeutic applications include ovarian cancer, fertility control, and the like. In a preferred embodiment, ricin conjugated anti-zona pellucida antibody is used to accelerate recruitment of primary follicles in the target ovary. Accelerated recruitment is useful for example, in contraceptive methods which effect later stage, e.g., tertiary, follicles. The anti-zona pellucida antibody specifically delivers the cytotoxic agent, ricin to the ovary. Ricin treatment results in loss of large follicles from the ovary (eg, tertiary follicles) and also results in recruitment of follicles (e.g., primordial follicles) into a growing pool where they may be acted upon by fertility agents. The immunochemical agents of the present invention may be administered to a patient by known conventional methods, e.g., injection, inhalation, or modified for oral delivery. Preferably, the agent is administered parenterally, i.e., intravenously, intraperitoneally, or the like. The agent is prepared in a pharmaceutically acceptable carrier, for example, in an aqueous medium such as water, buffer, saline, glycine, or an oil based carrier, as appropriate for the specific antibody-agent couple. A therapeutically effective dose, i.e., one that will eliminate, reduce, or retard the growth of the patient's tumor or one that will induce a desired fertility effect is administered. The therapeutically effective dose and dose regimen will vary according to specific characteristics of the patient, the therapeutic indication, and the nature of the therapeutic agent. The dose and regimen may be calculated by the treating physician according to acceptable procedures known in the therapeutic field. In general, a therapeutically effective dose will be less than the calculated LD 50 of the therapeutic agent as calculated for the weight of the patient. The amount of the agent typically will be in the range of about 0.01 to 100 mg/kg body weight and preferably between 0.01 and 10 mg/kg. The invention may be better understood by reference to the following examples: EXAMPLE 1 PREPARATION OF ANTI-ZONA PELLUCIDA ANTIBODIES Anti-zona pellucida antibodies against heat solubilized dog zona pellucida (HSDZ) were prepared generally following the procedures described by Dunbar et al., (Biochemistry. 19:356-365, 1980), except that ganged razor blades were used to mince the ovaries. Rabbits were immunized with a mixture of HSDZ (250 μg) and MDP (250 μg) . Two additional boosts followed at approximately three week intervals. The resultant rabbit serum was next used for IgG fractionation. Following the instructions provided with a Zymed Protein A column, an IgG fraction was collected. The collected anti-sera was incubated for one hour on the Protein A column. The column was then washed four times with PBS, IgG was eluted using 0.1 M acidic acid, pH 2.8, and buffered immediately with 1 M tris, pH 9.5. The collected samples were then dialyzed extensively against PBS, pH 7.2. The ELISA titer of the IgG fraction was greater than 64 K against HSDZ. EXAMPLE 2 ANTI ZP ANTIBODY-RICIN A CONJUGATE Antibodies produced as described for Example 1 were conjugated to deglycosolated Ricin chain A by Inland Laboratories, Dallas, Texas, following the procedures described in R. Fulton et al., Cancer Research, 48:2618-2625, 1989. The conjugated antibody was subsequently used in animal studies. Ricin-conjugated antibody (1 mg) prepared aε described above was placed in 0.5 ml PBS. The solution was then injected intravenously into a seven month old female dog. For the first 48 hours, the injected dog exhibited elevated temperature, lethargy, and some vomiting, but then returned to normal health. Seven days post-injection, the dog was unilaterally ovariectomized. The excised ovary was sectioned and stained for histological analysis. Analysis of stained sections of the ovary indicated accelerated atresia of tertiary follicles within the treated ovary as compared to control, untreated ovaries. Surprisingly, recruitment of primary and secondary follicles was accelerated in the treated ovary as compared to the controls. Control and treated histological sections are shown in Figures 1-9. Figures 1-4 show sections of control, untreated ovaries at 7.5, 10, 30, and 100 times magnification, respectively. Figures 5-8 show sections of treated ovaries at 7.5, 10, 30, and 100 times magnification, respectively. EXAMPLE 3 ^I-ANTI-ZONA PELLUCIDA ANTIBODIES Antibodies were produced against heat solubilized cat zona pellucida (HSCZ) following the procedures described for Example 1. Affinity purified rabbit anti-HSCZ IgG waε iodinated by Dr. R. Kittoke, University of Nebraεka, uεing the Iodogen method. A molar ratio of 2:1 125 I:protein was uεed. Reaction products were separated on a Bio-Gel P-60 column and the iodinated protein was eluted in the void volume. Approximately 124 million DPM of radioactivity waε associated with 15 μg of recovered protein. The radiolabeled protein (2.95 μg, 10.99 microcuries) in 0.59 ml PBS containing 0.1% gelatin waε injected intravenously into a female cat. The animal waε also injected with 50 μg of sodium Iodide in 0.5 ml PBS. The amount of sodium Iodicle was 10,000 timeε the amount of 125 I in the labeled protein, as calculated on a molar baεiε. Twenty-four hours post-injection the animal was euthanized and various tissues were removed. Tissues were homogenized and analyzed for radioactivity using a Micro-Medic gamma counter. As shown in Table 1, within 24 hours the amount of radioactivity within the ovary (CPM/g) was four times that of other reproductive tissues.

Claims: WE CLAIM : 1. A pharmaceutical composition comprising: an anti-zona pellucida antibody; a therapeutic agent conjugated to said anti- zona pellucida antibody, wherein said anti-zona pellucida antibody specifically bindε to ovarian tissue and thereby delivers the conjugated therapeutic agent to the ovary. 2. The pharmaceutical composition of claim 1, wherein said antibody is a polyclonal antibody. 3. The pharmaceutical composition of claim 1 wherein said antibody is a monoclonal antibody. 4. The pharmaceutical compoεition of claim 1 wherein said antibody is raised against a recombinant zona pellucida polypeptide. 5. The pharmaceutical composition of claim 1 wherein said therapeutic agent is a cytotoxin. 6. The pharmaceutical composition of claim 1 wherein said therapeutic agent is a radioisotope. 7. The pharmaceutical composition of claim 1, wherein said therapeutic agent iε a fertility control agent. 8. The pharmaceutical compoεition of claim 5, wherein said therapeutic agent is ricin. 9. A method of delivering a therapeutic agent to the ovary, said method comprising: conjugating a therapeutic agent to an antibody having specificity to ovarian zona pellucida; and administering to an individual a therapeutically effective amount of the antibody-agent conjugate for delivery of the therapeutic agent to the ovary. 10. The method of claim 9, wherein said therapeutic agent is a cytotoxin. 11. The method of claim 10, wherein said therapeutic agent is ricin A. 12. The method of claim 11, wherein said therapeutic agent is a radioisotope. 13. The method of claim 9, wherein said therapeutic agent is a fertility control agent. 14. A method for treating ovarian disease comprising administering to a patient in need of such treatment an effective therapeutic amount of the pharmaceutical composition of claim 1. 15. A method for inducing recruitment of ovarian follicles compriεing adminiεtering an effective dose of ricin conjugated to anti-zona pellucida antibody. 16. A method of inducing contraception in an animal comprising: inducing recruitment of primary ovarian follicles into tertiary follicles by administration of ricin-conjugated to anti-zona pellucida antibody; and inducing atresia of tertiary ovarian follicles.

Inventors: BOWMAN DAVID, HARRIS JEFFREY, PODOLSKI JOSEPH S, BOWMAN, DAVID, HARRIS, JEFFREY, PODOLSKI, JOSEPH, S.
Applicants: ZONAGEN INC, ZONAGEN, INCORPORATED
Application year: 1994
Main classification: A61K47
IPC codes: A61K39, A61K45, A61K47, A61K51, C07K16